This morning, I read back through
several of the STEC CAP newsletters
that the EMT has issued during the
course of our grand project, and it
sort of blows me away how many
truly remarkable accomplishments
have been made by our team. We
have improved detection methods
for STECs, we better understand
where these non-O157 STECs hide
out in production units and how
prevalent they are across different
cattle populations, we continue to
investigate the virulence profiles of
various STEC serogroups isolated
from cattle and how they might

Randy Phebus

H

appy spring to all of you, our
STEC CAP family and friends.
I sit today in my beloved
mancave enjoying the gorgeous
Kansas sunshine and thinking about
the myriad of accomplishments
we have made related to the STEC
CAP grant from back in 2010
when a small group of us began
the torturous endeavor of writing
the NIFA AFRI grant and pulling
the team together, to today where
we are embarking on our 6th year
of focused translational research,
education and outreach to improve
the safety of beef products, and
ultimately to improve public health.

impact food
safety and
public health,
our team of
economists
are involved
to gauge
cost-benefit
relationships of
different STEC
aspects in the
production
to consumption chain, we have
validated many intervention
technologies in lab studies and in fullscale production facility scenarios,
we have addressed many consumerlevel food safety risks and gained
a better understanding of human
behavior related to food safety, and
our robust Quantitative Microbial
Risk Assessment model continues
to evolve. We have trained a huge
number of graduate students who
will go on to accomplish great things
in food safety. We are nearing our
100th STEC-STEP internship being
filled, which was the achievement
benchmark set in our original grant
proposal. These interns have done
tremendous work to add to the
overall output and outcomes of all
five STEC CAP grant objectives.
...continued page 4
STEC CAP Team
126D VBS
Lincoln, NE 68905
PHONE: 402-472-8564
FAX: 402-472-9690

stecbeefsafety.org

Rapid-testing research
targets O serogroups

F

or the past three years,
rapid testing of Shiga
toxin-producing Escherichia
coli (STEC), one of the causes of
foodborne illnesses, has been
one focus of the Coordinated
Agricultural Program (CAP)
grant from the U.S. Department
of Agriculture (USDA). CAP
grant researchers especially are
developing and testing detection
methods for the several STEC
pathogens, including O26, O45,
O103, O104, O111, O121, O145
and O157, in the beef industry.
One such DNA-based detection
method is multiplex oligonucleotide
ligation-PCR (MOL-PCR), of which
CAP grant researchers Travis
Wood, a graduate student at the
University of New Mexico, and
his advisor, Alina Deshpande, a
scientist at Los Alamos National
Laboratory, led the development
and validation. The assay is now in
the process of independent testing
by another CAP grant researcher.
MOL-PCR can rapidly screen
samples concurrently for several
STEC O serogroups and three
major virulence factors (genes
for two Shiga toxins and intimin).
Inside this issue

STEC CAP News
In turn, the CAP grant is using a
waveguide-based optical biosensor,
with development led by Loreen
Stromberg, currently a postdoctoral
research associate at Iowa State
University, and her advisor, Harshini
Mukundan, a scientist at the Los
Alamos National Laboratory, to
detect the binding of a key surface
molecule, lipopolysaccharide,
which is the outer part of the
membrane of the E. coli. CAP grant
researchers use antibodies to detect
the lipopolysaccharide in a very
sensitive method that eliminates a
lot of background.

...continued from page 1

Rapid-testing research targets O
An isolate would have to be positive for one of the targeted O serogroups, as well as a
Shiga toxin and intimidating, to be classified as an adulterant.

According to the USDA Food Safety and Inspection
Service (FSIS), to be classified as an adulterant, an
isolate would have to be positive for one of the targeted
O serogroups as well as a Shiga toxin and intimin.
The MOL-PCR STEC assay that has been developed
is a screening test, and CAP grant researchers have
developed it to run on DNA from culture. Beginning with
the culture material, MOL-PCR produces results in the
same day. Beginning with a ground beef sample, results
are possible in two days.
Besides independent lab testing of the MOL-PCR
screening test, CAP grant researchers are working on
a MOL-PCR characterization assay. This assay detects
the seven non-O157 STEC that are adulterants, but it
is looking at a different gene than the first MOL-PCR.
In essence, the characterization assay is testing for
individual genes representing each of the seven types
of adulterants that would be further confirmation that
particular sample has the adulterant in it, explains Rod
Moxley, a professor at the School of Veterinary Medicine
and Biomedical Sciences at the University of Nebraska–
Lincoln and project director of the CAP grant. CAP grant
researchers are looking at a variation of one gene, called
a single nucleotide polymorphism (SNP). One individual
nucleic acid substitution in that gene makes it unique, and
the types of E. coli that are adulterants would have that
kind of substitution.
“It’s targeting a sequence that’s very typical for a
particular, let’s say O1O3, O111 or O26, but it would be
a second type of MOL-PCR assay that would be called a
characterization assay,” Moxley explains. “We could do it
on that same sample that’s been screened, and then come
up with more additional confirmatory evidence that the
adulterant is there.”
While several PCR assays that can search for multiple
genes at a time exist, MOL-PCR is rapid and high

STEC CAP News

throughput, translating to a lot more samples being
run through, in comparison to end-point PCR and
quantitative real-time PCR (qPCR). MOL-PCR also is
relatively inexpensive. In addition, while MOL-PCR has
been developed to test for 11 markers, it is adaptable
and can potentially go up to 50 at a time.
“What that means is that you can get a tremendous
amount of information about a sample because you are
looking at many, many different targets at once,” Moxley
explains. “The more evidence you have, the greater the
likelihood of something being positive. You also can easily
add more genetic markers to this assay that you already
developed. Instead of starting all over again, you can add
things into the mix, which is a big advantage.”
Outside of the CAP grant research, another rapid
technology researchers are studying involves droplet
digital PCR (ddPCR). Like qPCR, this method allows
for quantification of STEC target genes; however, its
researchers say ddPCR has a couple of advantages. First,
ddPCR yields accurate absolute quantification without
the need to rely on a standard curve. This is important
because the construction of a standard curve requires
accurately quantified template DNA, which might be
difficult to obtain. Second, ddPCR is less prone to inhibitors
of DNA polymerase, e.g., bile salts, than qPCR.

Detecting biomarkers

CAP grant researchers also have been working to address
the USDA request for development of biosensors, which
in essence would detect biological phenomenon. For
example, this could be a binding of part of a pathogen to
a surface or to a receptor that would normally be present
on a cell or body.
“The way pathogens infect you is by binding to
something that allows them to get a foothold on the
surface and then establish a connection that allows
them to grow,” Moxley says.

“It works by detecting key molecules
in a pathogen by an ability to bind
to the surface,” Moxley explains.
“Then antibodies are used that
are specific for that molecule that
have a fluorescent label on them,
and that florescent light can be
detected. Then another key part of
the way this system works is that
light fades out a short distance
above the surface where the
pathogen is bound. The further
you get, and we are talking in an
extremely minute space here above
the membrane, that light completely
disappears. That really eliminates

a lot of artificial signal, which is
important in the system.”
This waveguide biosensor assay is
very different from the MOL-PCR
or any of the DNA-based methods.
“This is detecting an actual part of
the bacteria, an outer membrane,
as opposed to its DNA,” Moxley
says. “There aren’t many places
around the country that are
developing methods for detecting
non-DNA targets.”

From Student to Scientist

O

riginally from the Gem State of Idaho, my
journey to work with the best food safety
professionals in the country brought me to the
Midwest. When I think about the first time I
walked into the Food Safety and Defense Laboratory
at Kansas State University, I had no idea the level or
the number of meaningful research projects I would
be exposed to. With the support of Dr. Randall Phebus
and the STEC CAP grant, I began working towards a
Master’s degree in August 2014.
My thesis project consisted of a two-part evaluation of a
novel chlorinated nanobubble antimicrobial technology
for use in proprietary beef processing developments.
This was especially exciting as nanobubble technology
is relatively new to the food industry, but proved to be
equally challenging due to its minimally-researched
state. Fortunately, this served as an excellent opportunity
to team up with industry partners to evaluate the
antimicrobial properties of chlorinated nanobubble
waters against the foodborne pathogens STEC and
Salmonella, and non-pathogenic E. coli surrogates, in
pure and organically-loaded solution scenarios on a
benchtop level. The second stage of this research project
consisted of conducting a month-long in-plant validation
of a recirculating chlorinated nanobubble solution within
a beef trim processing plant—an enormous task that
could not have been completed without expert help
(specifically Drs. John Luchansky and Anna Porto-Fett
and graduate student Nick Sevart for sacrificing a month
of their lives to help me) and extensive planning. One
huge benefit of the STEC CAP grant is the ability to work
with and bounce ideas off of seasoned researchers from
other institutions—my own research could not have been
successful without many pairs of eyes and thoughtful
input. Learning to collaborate with industry members,

academic researchers, and accounting
for regulatory requirements was
difficult, but rewarding once finished
with the project.
Before working with STEC CAP, I
felt my understanding of the beef
industry to be fairly extensive due to
my previous internship experiences
at Washington Beef, LLC in Toppenish, WA and my
personal background of raising livestock on a family
farm; but, I had no idea the level of scientific depth
the industry utilized to mitigate foodborne pathogens
and evaluate antimicrobial interventions. Working with
Dr. Phebus and my lab mates on several of the STEC
CAP’s Objective 3 projects really broadened my depth
of understanding and deepened my appreciation for
academic research.
I can honestly say that the experiences I gained working
with STEC CAP associated projects, specifically my
own thesis project, developed my professional and
personal skills tremendously. The grant provided real-life
multidisciplinary collaborative research opportunities,
something that has helped me transition smoothly into
a career as a scientist in industry. My ability to now
confidently conduct research and critically evaluate and
solve problems will forever benefit my future endeavors,
a feeling I hope other STEC CAP students will experience
in their time working with grant-assisted projects.
In December 2016, I walked away from my STEC CAP
supported project with a Master’s of Science in Food
Science from Kansas State University and a job as an
R&D Concept Developer with Boar’s Head Brand in
Holland, MI.

STEC CAP News

STEC CAP News

...Directors Update ...continued from page 1
STEC CAP NATION … YOU HAVE
PERFORMED WELL … thus far!
A significant amount of work remains
to be completed by every person
affiliated with the STEC CAP grant,
as we get closer to the end. We will
characterize this work as
1) finishing/expanding current
projects, of which there are many;
2) starting and completing several
new projects; 3) reporting and
documenting past, current, and
future projects and grant-related
achievements in a timely manner;
4) comprehensively summarizing
the accomplishments of our work
towards our originally stated goals;
and 5) efficiently reassessing all
outstanding budgets where dollars
remain unspent to ensure the best
and complete use of our remaining
resources. The EMT, along with our
grant’s financial/business advisors at
UNL, will be devoting a huge effort
to make sure that all of the above
happen smoothly and completely.
We ask all of you to assess the status
of your own STEC CAP obligations
over the next couple of weeks to
align your unspent budgets with your

research, education and outreach
activities through this no-cost
extension period ending November
30, 2017. Objective team leaders…
we ask you to help us across your
respective teams to accomplish
this evaluation and propose ideas
for best achieving remaining goals
with any unspent funds. By the
end of May 2017, the EMT will be
making significant programmatic
and budgetary decisions related to
closing out the grant. We would like
to hear from all co-PIs before these
decisions are made.
Now… an announcement that
makes the EMT a little nervous to
make. Through recent discussions
with our Program Leader at NIFA,
it may be possible that we can
make the case for a 7th year nocost extension to finish off key
elements of the grant’s deliverables.
However, there has to be very good
justification for this extension and
NIFA has to approve it. The EMT
is encouraging co-PIs to target
completion of projects by our 6th
year deadline of November 30,
2017, but we want you to almost

Mark your calendars now for the STEC CAP IAFP Breakfast!

The annual STEC CAP IAFP breakfast will be held Tuesday, July 11th at the Downtown
Embassy Suites Tampa, FL. Continental breakfast at 7:00 AM with meeting to follow.

immediately inform us if you feel
you have a justifiable need (and
remaining funding) to extend your
STEC CAP work into 2018.
Dr. Moxley will put together a
formal 7th year no-cost extension
request with detailed justification
and submit it to NIFA shortly after
our summer STEC CAP annual
conference (Dr. Walls has asked for
it to be submitted in August 2017).
As you evaluate your needs in this
regards, please understand that if
we are awarded the 7th year nocost extension, UNL will require all
subcontractors to have completed
their projects and any expenditures
(including salaries/student stipends)
by August ?? (JILL), 2018 (no
extensions or exceptions).
The most obvious indication of our
STEC CAP efforts is our output of
refereed journal manuscripts. In
case you haven’t noticed, we have
a beautiful and valuable website
that is one of our best informational
avenues to the outside world.
Under a tab called Researchers
in Action (http://stecbeefsafety.
org/researchers-action),
you will see a comprehensive
listing of published and in-press
manuscripts from our grant
activities, with links to most of
these articles. Currently, we sit at
80 peer-reviewed manuscripts.
A significant number of projects
have been completed and
manuscripts are in various stages
of development. The EMT and
our NIFA program leader are
sending out this request, or we
will call it a challenge…LET’S
HAVE A MINIMUM OF 100
MANUSCRIPTS SUBMITTED
TO JOURNALS BEFORE OUR
JUNE 13-15, 2017 ANNUAL
CONFERENCE! Please prioritize
getting these manuscripts
submitted so that we can show
our great productivity as we
request the 7th year no-cost

EARLY
REGISTRATION
PRICING
UNTIL APRIL 30TH
Students can register for FREE
See complete agenda online

http://stecbeefsafety.org/2017-steccap-annual-conference
extension paperwork. Don’t forget to fill out and
submit the short Manuscript Documentation Form
(http://stecbeefsafety.org/points-board) to the
EMT immediately before you send these manuscripts
to journals. While you’re at this area of our website,
click on the Points-on-the-Board link and record all of
the great things you’re group has done so we can get
these into our agency reporting.
The STEC CAP grant continues to receive frequent
and positive media coverage, and our team has
been busy on many fronts spreading the word of
our contributions towards beef/food safety. Dr.
Moxley visited K-State last month and presented
an outstanding keynote seminar on virulence
characteristics of STEC to our College of Veterinary
Medicine. I gave a keynote address at Oklahoma
State University in February that focused on validation
of interventions (http://fapc.biz/workshops/2017research-symposium/researchsymposium) to
control pathogens in foods (skip to the 47 min mark
for STEC CAP piece), and was fortunate to be invited
to Texas A&M also to speak to food/meat science
students and faculty regarding our STEC CAP
accomplishments. Just this week, the faculty retirees
social club from the K-State Colleges of Ag and Vet
Med invited me to speak to them about our STEC
CAP’s educational and outreach efforts. What an
energetic group they were! Please, let the EMT know
when you make presentations or have significant
outreach communications (and report it in the POBs)
so we can thank you and promote your effort.
You may want to check out the additional national
coverage we’ve received through these links:
http://www.stopfoodborneillness.org/candcrodney-moxley-december2016/

http://www.hpj.com/ag_news/dodge-city-sharesin-usda-grant/article_562cf5c0-a29b-11e6-8a9193b6545b0706.html
http://www.leavenworthtimes.com/news/20161025/
schools-to-benefit-from-grant
http://www.provisioneronline.com/articles/104052rapid-testing-research-targets-o-serogroups
http://news.unl.edu/newsrooms/today/article/e-coliproject-generates-new-detection-control-methods/
It’s exciting to think about our upcoming STEC CAP annual
conference that will occur June 13-15, 2017 in Lincoln, NE,
again at the Embassy Suites. This is our fifth one! This time
the conference will be open to the public with a proper
registration. We will have some very high-profile speakers
from both within and outside of the STEC CAP family.
Encourage your students and colleagues to log in and
register soon (http://stecbeefsafety.org/2017-stec-capannual-conference).
On behalf of my EMT comrades, thank you for your
continued professionalism, great science, and passion for
addressing a significant challenge to our food system…
Shiga toxigenic E. coli in beef products. Please re-energize
yourselves to help us sprint towards the finish line with
this landmark grant, and let’s look for ways that our 6-7
year group effort can synergize to go after future support
opportunities. The EMT is at your service, so feel free at
anytime to contact us with ideas, wishes, concerns, and
usable information.
OK…I’m now back to enjoying my Spring Break (not really,
we have an EMT teleconference in 15 min. Ughhhh!
by Randy Phebus (phebus@ksu.edu)

STEC CAP News

STEC CAP News
industry. Local employers understand what type of
employees they need and Dodge City schools can help
guide students to those careers.
Employers and educators can serve as mentors, she
said. Through mentoring experience a student may find
the career path is not what he or she wanted to do,
which can also be a productive experience.
Kyle Longacre, an educator from Souderton, said
the award and program is designed for the entire
community.
“It will help our children be better prepared for
the world,” said Dodge City Superintendent Alan
Cunningham.

Jason Ellis, an associate professor in the College of
Agriculture at K-State, said the program represents an
opportunity to build economic value and development
from within a community. Regardless of where he
travels in the Midwest, the idea of providing jobs
and opportunities for youth, is a common theme
in which community leaders acknowledge lost
opportunities when young people leave. Development
and redevelopment are important and growing future
employees and businesses from within a community is a
successful and sustaining formula.
Business leaders commended the school district for
pursing the grant and for looking for ways to partner
with them.

Dodge City Receives STEC Grant

D

odge City High School hosted representatives
from Pennsylvania’s Souderton High School,
Kansas State University, as well as the University
of Nebraska-Lincoln on Oct. 27 and 28 for the
purpose of exploring ways to more effectively educate
and prepare students for future careers.
The U.S. Department of Agriculture’s National Institute
of Food and Agriculture funded the STEC Coordinated
Agriculture Project goal is to reduce the public health
risk and number of cases of Shiga
toxin-producing Escherichia-coli
infections in the population. As part
of the event on Oct. 28, Dodge
City High School was awarded a
$100,000 from USDA. Dodge City
was one of 18 institutions sharing
in a $2.5 million grant program.
“The STEC Cap partnered with
Pathway 360 Degrees to increase
awareness about the potential
dangers of Shiga toxin-producing Escherichia-coli in
beef while giving students a powerful venue to expose
them to career opportunities and new possibilities,”
according to a news release from Dodge City schools.
Finding ways to develop careers in food science and
related fields can occur with proper preparation that
can start at the secondary level before a student even
reaches college, representatives said.
The Pathway 360 program was developed by Souderton
High School.

“Through the program, students critically think about
potential career choices, which allows them to make
better decisions about their professional paths,” stated
Rod Moxley, STEC Cap grant director.
“Pathway 360 can help educate students on not just
food safety, but all types of agricultural careers that
are continually increasing, offering students real world
experience,” according to Jill Hochstein, STEC CAP
project manager. “We are hopeful that the program
will continue to grow and expand to more areas across
the country. Souderton’s Pathway 360 program is an
inspiration to the growing career needs of our young
people and the STEC CAP Grant is proud to be a
partner in this initiative.”
Dodge City High School is working in conjunction with
the STEC Cap Project.
“We’re currently in the research and planning phase
to determine how we might implement the key
components of the Pathway 360 program to enhance
the educational experiences of our students,” Dodge
City High School Principal Jacque Feist said. “This fits
nicely with the individual plans of study all our students
are currently building as part of the New Vision for
Education in Kansas.”
During a public presentation, members of the
business community, school board members and local
government officials, as well as educators from the
schools and local community college. Feist said Dodge
City was a natural place for such a project because
agriculture and livestock production is the region’s top

Check us out on the Web! Visit us at: www.stecbeefsafety.org
Subscribe to the listserv. Send an email to: listserv@unl.edu In the message field: subscribe stecbeefsafety
This project was supported by Agriculture and Food Research Initiative Grant No. 2012-68003-30155 from the USDA National Institute of Food and Agriculture,
Prevention, Detection and Control of Shiga Toxin Producing Escherichia coli (STEC) from Pre-Harvest Through Consumption of Beef Products Program –A4101.